title: P2X4 receptors interact with both P2X2 and P2X7 receptors in the form of homotrimers.
abstract: OBJECTIVE
  The P2X receptor family consists of seven subunit types - P2X1-P2X7. All but P2X6 are able to assemble as homotrimers. In addition, various subunit permutations have been reported to form heterotrimers. Evidence for heterotrimer formation includes co-localization, co-immunoprecipitation and the generation of receptors with novel functional properties; however, direct structural evidence for heteromer formation, such as chemical cross-linking and single-molecule imaging, is available in only a few cases. Here we examined the nature of the interaction between two pairs of subunits - P2X2 and P2X4, and P2X4 and P2X7.
  
  
  METHODS
  We used several experimental approaches, including in situ proximity ligation, co-immunoprecipitation, co-isolation on affinity beads, chemical cross-linking and atomic force microscopy (AFM) imaging.
  
  
  RESULTS
  Both pairs of subunits co-localize upon co-transfection, interact intimately within cells, and can be co-immunoprecipitated and co-isolated from cell extracts. Despite this, chemical cross-linking failed to show evidence for heteromer formation. AFM imaging of isolated receptors showed that all three subunits had the propensity to form receptor dimers. This self-association is likely to account for the observed close interaction between the subunit pairs, in the absence of true heteromer formation.
  
  
  CONCLUSIONS
  We conclude that both pairs of receptors interact in the form of distinct homomers. We urge caution in the interpretation of biochemical evidence indicating heteromer formation in other cases.
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge, UK.
authors.forename: L S
authors.initials: LS
authors.lastname: Antonio
authors.forename: A P
authors.initials: AP
authors.lastname: Stewart
authors.forename: X J
authors.initials: XJ
authors.lastname: Xu
authors.forename: W A
authors.initials: WA
authors.lastname: Varanda
authors.forename: R D
authors.initials: RD
authors.lastname: Murrell-Lagnado
authors.forename: J M
authors.initials: JM
authors.lastname: Edwardson
mesh_terms: D018929:Cell Culture Techniques; D003432:Cross-Linking Reagents; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D018625:Microscopy, Atomic Force; D055503:Protein Multimerization; D021122:Protein Subunits; D058476:Receptors, Purinergic P2X2 / Q000737:chemistry* / Q000235:genetics; D058848:Receptors, Purinergic P2X4 / Q000737:chemistry* / Q000235:genetics; D058486:Receptors, Purinergic P2X7 / Q000737:chemistry* / Q000235:genetics; D014162:Transfection
chemical_list: D003432:Cross-Linking Reagents; D021122:Protein Subunits; D058476:Receptors, Purinergic P2X2; D058848:Receptors, Purinergic P2X4; D058486:Receptors, Purinergic P2X7